Research ArticleExperimental Studies

In Vivo MRI of Intraspinally Injected SPIO-labelled Human CD34+ Cells in a Transgenic Mouse Model of ALS

S. WILLENBROCK, S. KNIPPENBERG, M. MEIER, R. HASS, P. WEFSTAEDT, I. NOLTE, H. MURUA ESCOBAR and S. PETRI
In Vivo January 2012, 26 (1) 31-38;

Abstract

Background/Aim: Administration of stem cells is a promising novel approach for treatment of neurodegenerative diseases. For in vivo monitoring of transplanted cells, non-invasive imaging modalities are needed. In this study we determined the tracking efficiency of a superparamagnetic iron oxide (SPIO)-labelled canine cell line (MTH53A) in vitro as well as the human CD34+ umbilical cord blood stem cells (hUCBCs) in vitro and in vivo efficiency by magnetic resonance imaging (MRI). Materials and Methods: SPIO-labelled MTH53A cells and hUCBCs were scanned in agar gel phantoms at 1.0 T or 7.0 T. For in vivo detection, 100,000 labelled hUCBCs were injected into the spinal cord of a transgenic amyotrophic lateral sclerosis (ALS) mouse and scanned at 7.0 T. Results: In vitro, 100,000 MTH53A cells and 250,000 hUCBCs were visible at 1.0 T. Scanning with 7.0 T revealed 25,000 detectable MTH53A cells. In vivo, 7.0 T MRI showed clear signals of 100,000 implanted cells. Conclusion: MRI combined with SPIO nanoparticles provides valuable potential for non-invasive, non-toxic in vivo tracking of cells implanted into the spinal cord.

Cellular therapies represent a novel approach for the treatment of a variety of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). ALS is the most common adult motor neuron disorder characterized by progressive degeneration of motor neurons in the brainstem, the primary motor cortex and the spinal cord, with a mean survival of about three to five years (1). To date, the causes of ALS are still undefined and effective therapeutic strategies are lacking (2-4). While the majority of ALS cases (>90%) are sporadic, mutations in the superoxide dismutase I (SODI) gene were detected as a genetic cause of familial ALS (5). This led to the generation of the most commonly used and best characterized mouse model of ALS. SOD1-G93A mice overexpress human mutant SOD1 closely mimicing human ALS (6, 7).

Transplantation of stem or progenitor cells is being evaluated with the purpose of either replacing lost or dysfunctional neurons, or creating a neuroprotective environment for degenerating neurons, e.g. by secretion of trophic factors and/or anti-inflammatory effects (8, 9). In spite of a large number of in vivo studies in transgenic animal models, it is still not sufficiently understood how many cells transplanted into the central nervous system migrate and differentiate. Moreover, for translation into clinical trials, the development of safe in vivo imaging methods to monitor the fate of transplanted cells is urgently needed (8, 9).

Different types of cells, such as embryonic stem cells, neural stem cells, bone marrow cells and umbilical cord blood cells, are suitable for cellular therapy of neurodegenerative diseases (9). Among these cell types, adult stem cells from human umbilical cord blood (hUCBCs) are discussed as being preferable due to their non-fetal origin, ethical innocuousness, low immunogenic potential, and easy availability (10). While bone marrow-derived cells and hUCBCs have already been shown to have therapeutic potential in the mouse model of ALS (11-13) and have even been applied in some human ALS patients (14, 15), suitable non-invasive, non-toxic in vivo imaging methods to better understand the fate and migration of transplanted cells are lacking. In this context, magnetic resonance imaging (MRI) appears to be a promising clinically transferable tool, with high spatial resolution, a long effective imaging window, and fine signal intensity (16). In order to detect transplanted cells by MRI, the cells must be labelled with an effective contrast agent. Due to their low toxicity and high biocompatibility, different types of superparamagnetic iron oxide (SPIO) nanoparticles with strong T2* specific signal extinctions are widely used for labelling of different stem cell types (16-22).

Human umbilical cord mesenchymal stem cells (hUC-MSCs) labelled with the FDA-approved SPIO-particle Feridex have been assessed in vitro and in vivo after transplantation into the dorsal spinal cord of Sprague-Dawley rats by MRI using a 1.5 T scanner in a study by Hu et al. (23). A study by Delalat et al. (24) examined the in vivo migratory capacity of hUCBCs and human mesenchymal stem cells (MSCs) isolated from bone marrow labelled with the FDA-approved SPIO-contrast agent Endorem. These cell populations were transplanted into Balb/c mice by tail vein injection and detected by histopathology using Prussian blue (PB) staining, but not by MRI (24). Endorem is widely used as MRI contrast agent and can be easily incorporated by endocytosis. In contrast to other SPIO particles, the uptake of Endorem does not require transfection agents, which may reduce the viability of cells (25).

For the first time, we have specifically evaluated the tracking efficiency of Endorem-labelled CD34+ hUCBCs in vitro and in vivo by MRI after intraspinal injection in a transgenic mouse model of ALS.

Materials and Methods

Cultivation of canine MTH53A cells. The canine mammary epithelial cell line MTH53A was chosen due to its unlimited availability to perform the ‘proof of concept’ for determination of the lowest detectable SPIO-labelled cell numbers via 1.0 T and 7.0 T MRI ex vivo in an agar gel matrix. The MTH53A canine mammary cell line, derived from healthy canine mammary epithelial tissue, was established at the Centre for Human Genetics, University of Bremen, Bremen, Germany. MTH53A cells were grown at 37°C and 5% CO2 in 199 medium (Gibco, Karlsruhe, Gemany) supplemented with 10% heat-inactivated foetal calf serum (PAA Laboratories GmbH, Coelbe, Germany), 200 U/ml penicillin and 200 ng/ml streptomycin (Biochrom AG, Berlin, Germany).

Isolation and ex vivo expansion of CD34+ hUCBCs. Isolation of CD34+ cells was performed by magnetic antibody cell sorting (MACS) from human umbilical cord blood. Thus, cord blood was obtained from healthy pregnant women with non-complicated pregnancies at spontaneous term-deliveries (38-40 weeks of gestation) or by Caesarean section after informed written consent, respectively, as approved by the Institutional Review Board, project #3037 on June 17th, 2006. The cord blood was immediately transferred into EDTA-containing monovettes on ice and separated via Ficoll density gradient centrifugation. Following removal of the lymphocyte interphase, further separation was performed via the CD34+-associated MACS technology according to the manufacturer's instructions (Miltenyi GmbH, Bergisch Gladbach, Germany). The isolated population was analyzed for appropriate CD34+ hematopoietic stem cell enrichment by flow cytometry. Thereafter, the separated primary stem cell population was cultured and expanded in the presence of Iscove's Modified Dulbecco's Medium (IMDM; Biochrom AG, Berlin, Germany), 10% FCS (PAA Laboratories GmbH, Coelbe, Germany), 1% Pen/Strep (Biochrom AG), Stem Cell Factor (SCF), Interleukin-3 (IL-3), Interleukin-6 (IL-6), FMS-like tyrosine kinase 3 (FLT-3) (all growth factors and cytokines were purchased from PeproTech GmbH, Hamburg, Germany) at 5% CO2 and 37°C for 7 to 14 days with a medium change every third day. Before surgery, the expanded cells (hereafter termed hUCBCs) were washed twice with a 0.9% (w/v) sodium chloride solution and resuspended at a density of 100,000 cells/μl.

In vitro SPIO cell labelling. Cell labelling was performed with the commercially available SPIO suspension ENDOREM® (Guerbet S.A., Roissy, France). This infusion suspension contains particles with an approximate size of 80 to 150 nm and has a total iron content of 11.2 mg/ml.

A total of 5×106 MTH53A cells or CD34+ hUCBCs was seeded in a 25 cm2 cell culture flask with 5 ml of the respective cultivation medium. For labelling, 41.15 μl SPIO suspension (=130 pg iron oxide nanoparticles/cell) was added to the seeded cells followed by an overnight incubation at 37°C in 5% CO2. After SPIO incubation, the viability of the labelled cells and unlabelled cells as controls was assessed by trypan blue staining.

Prussian blue staining. After SPIO labelling, cells were fixed with 4% paraformaldehyde, washed with 1× Phosphate Buffered Saline (PBS; Biochrom AG, Berlin, Germany) and incubated with a 1:1 solution of 5% potassium ferrocyanide and 5% hydrochloric acid for 30 min. The ferric iron (iron(III) oxide; Fe2O3) of the intracellular SPIO particles reacts with potassium ferrocyanide to form ferric ferrocyanide (=Prussian blue), a water-insoluble, blue precipitate. A final washing step with 1× PBS was performed before visualization of the internalized particles was carried out under light microscopy.

Agar gel phantom construction. For ex vivo MRI detection of SPIO-labelled cells, 250 ml of a hand-warm bubble-free 1% agar (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) solution (in dH2O) were prepared and put into an empty pipette tip box (Greiner Bio-One, Frickenhausen, Germany). For generation of sample wells, an unskirted 96-well PCR plate (Eppendorf AG, Hamburg, Germany) was placed onto the surface of the liquid agar solution. After polymerisation of the agar, the 96 well-plate was removed and the sample wells were ready for cell loading.

The SPIO-labelled cells were trypsinized after incubation with SPIOs and the cell number was determined. Defined numbers of cells were aliquoted into 1.5 ml cups (Eppendorf AG, Hamburg, Germany) as indicated in the results. The aliquoted cells were centrifuged for 10 min at 1,000 rpm (room temperature), the supernatant was discarded and the pellet was resuspended in 30 μl of hand-warm 4% gelatine (AppliChem, Darmstadt, Germany)/dH2O solution. The cell-gelatine mixture was pipetted into the wells of the agar gel phantom and air bubbles were removed. The phantom was cooled at 4°C until the gelatine was solidified. As controls, additionally to unlabelled cells, 1.0 μl or 1.5 μl SPIO solution (1.0 T MRI and 7.0 T MRI, respectively) and 30 μl culture medium were prepared by mixing 30 μl of the 4% gelatine solution and loading the samples into the wells of the agar gel phantom. Finally, to embed the loaded samples evenly in the agar phantom, the top was covered with 1% agar gel solution. After polymerisation, the construct was stored at 4°C until MRI analysis.

In vivo experiment. In vivo imaging of SPIO-labelled hUCBCs was performed in a transgenic mutant SOD1-ALS mouse. The G93A-ALS mouse model (B6SJL-Tg(SOD1-G93A)1Gur/J) is bred and maintained at the animal facility of Hannover Medical School. The animal experiment in this study has been approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES, Oldenburg) (AZ 06/1128).

SPIO-labelled cells were trypsinised, washed twice in PBS (Biochrom AG, Berlin, Germany) and the cell density was adjusted to 100,000 cells/ml. A centrifugation step was performed at 1,000 rpm for 10 min, at room temperature, to pellet the cells and the supernatant was discarded. To remove residual cell culture medium, the cell pellet was resuspended in sterile 1× PBS (Biochrom AG, Berlin, Germany) and centrifuged again. After a second PBS washing step, the cell pellet was resuspended in an adequate volume of sterile PBS to prepare a solution of 100,000 cells/ml.

For surgery, the animal was anaesthetized by a combination of ketamine (0.1 ml/100 g, 100 mg/kg), xylazine 2% (0.01 ml/100g, 2 mg/kg) and midazolame (0.05 ml/100 g, 0.5 mg/kg), prepared under sterile conditions with 0.9% sodium chloride. Anaesthesia was administered intraperitoneally, adjusted to the body weight (0.1 ml/10 g) and lasted 60 min as controlled by the toe and eyelid reflex. An area of 3×2 cm on the back of the animal was disinfected and shaved. Eye ointment protected the eyes against dehydration. The animal was fixed in ventral position by tape and the skin was disclosed longitudinally. Laminectomy was performed at vertebral bodies Th12/L1 with sharp scissors to expose the spinal cord at level L1-L4. For better visibility, the backbone adjacent to the exposed spinal cord was fixed with a clamp, which again was fixed in a stereotactic frame. A Hamilton syringe with an elongated glass capillary on top (50-80 μm diameter) was used for injection. The syringe was filled with 0.9% sodium chloride before 1 μl stem cell solution (i.e. 100,000 cells in 0.9% sodium chloride) was administered into the lumbar region of the spinal cord. The syringe was clamped into the arm of the stereotactic frame and inserted 1 mm in the ventral direction from the dorsal surface of the spinal cord, followed by slow injection of the cells over a period of three minutes. After a period of an additional two minutes, the syringe was removed slowly to prevent reflux of inserted cells. After wound closure, the animal received a single dose of carprofene (5 mg/kg subcutaneously) and metamizole via drinking water (200 mg/kg/day) for 3 post-surgical days for analgesia.

In vitro MRI. The scans of the prepared agar gel phantom, loaded with SPIO-labelled cells and respective controls, were performed in a clinical whole-body MR imaging system at 1.0 T (Magnetom Expert, Siemens Healthcare, Erlangen, Germany) and a Bruker Pharmascan 70/16 7.0 T MR-tomograph for small laboratory animals (Bruker BioSpin MRI GmbH, Ettlingen, Germany).

For 1.0 T analysis, a T2*-weighted gradient echo flash 2-D sequence with a repetition time (TR) of 800 ms and an echo time (TE) of 26.0 ms, slice thickness of 2.0 mm, flip angle (FA) of 20°, field of view (FoV) of 201×230, 168×256 and two repetitions was used and the data analysis was carried out by dicomPACS version 5.2 (Oehm and Rehbein, Rostock, Germany).

To scan the agar gel phantom in the 7.0 T MRI, an 8 cm volume resonator designed for MRI analysis of rats was used. The agar gel phantom was trimmed with a scalpel to fit into the 8 cm volume resonator. The parameters for the 7.0 T scan were as follows: T2*-weighted flash 2D sequence, TR/TE=200/20, slice thickness 2.0 mm, FA=90°, one repetition.

Data were analysed using ImageJ, version 1.41 (NIH, Bethesda, MD, USA) extended by the Bruker Opener plugin version 2008/04/22 (Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany).

In vivo MRI. In vivo MRI was performed on a 7.0 T Bruker Pharmascan 70/16 (Bruker Biospin) equipped with a 6 cm volume resonator using Paravision 5.0. The mouse was anaesthetized with isoflurane during the MRI scans. The body temperature was kept at approximately 37°C using a temperature control unit (Small Animal Instruments, Stony Brook, NY, USA). A T2* multi-gradient echo (MGE) with the following parameters was used: TR=1500 ms, TE=9 ms, FA=30°, slice thickness=1 mm.

The G93A-ALS mouse was scanned at day 0 (injection day) and day 4 to detect the injected SPIO-labelled cells and to analyse if cell migration could be visualized.

Results

1.0 T and 7.0 T MRI scans of ex vivo agar gel phantoms. MTH53A cells were used for the proof of concept experiments. The 1.0 T MRI scan of SPIO-labelled canine MTH53A cells embedded in the agar gel phantom revealed a cell detection limit of 1×105 labelled cells due to SPIO-induced T2*-specific signal extinctions (Figure 1A). The respective negative controls (medium alone and unlabelled cells) showed no signal extinction and the positive controls with 1.0 and 1.5 μl Endorem® showed strong signal extinctions.

To examine the cellular detection limit at higher field strength, an agar gel phantom with SPIO-labelled MTH53A cells was scanned with 7.0 T MRI (Figure 1B). Medium and unlabelled cells as negative controls exhibited no signals. The positive control with 1.0 μl Endorem® demonstrated a strong T2*-specific signal extinction. The 7.0 T MRI scan revealed a cell detection limit of 2.5×104 labelled cells in comparison to the detection limit of 1×105 labelled cells at 1.0 T field strength.

1.0 T MRI analysis of SPIO-labelled CD34+ hUCBCs in the agar gel phantom detected a minimal limit of 2.5×105 labelled cells, while controls (medium and unlabelled cells) were undetectable (Figure 2). The positive controls (1.0 and 1.5 μl Endorem®) showed signal extinctions as observed in the agar gel phantom with SPIO-labelled MTH53A cells.

In vitro cell labelling, viability and Prussian blue staining. Trypan blue staining was performed directly after the respective labelling reaction. All labelled and unlabelled cells had a viability of nearly 98% (data not shown). After staining with Prussian blue, an accumulation of iron in the cytosol of SPIO labelled cells was detected, indicating that the cells were successfully labelled for MRI tracking (Figure 3A and B).

Figure 1.
Figure 1.

Ex vivo agar gel phantom detection of superparamagnetic iron oxide (SPIO) labelled canine MTH53A cells by 1.0 and 7.0 T magnet resonance imaging (MRI). A: 1.0 T MRI showing embedded numbers of MTH53A cells. IPC, Positive control (1.0 and 1.5 μl Endorem solution); NC1, cell culture medium; NC2, 1×104 unlabelled cells; NC3, 7×105 unlabelled cells. Detection limit of SPIO-labelled cells was determined as 1×105 by 1.0 T MRI. The negative controls NC1, NC2 and NC3 show no signals. The positive control IPC shows strong signal extinction. B: 7.0 T MRI showing embedded numbers of MTH53A cells. IPC, Positive control (1.0 μl Endorem solution); NC1, cell culture medium; NC2, 5×105 unlabelled cells. The controls show the expected signals corresponding to Figure 2A. Detection limit of SPIO-labelled cells was determined as 2.5×104 by 7.0 T MRI.

In vivo 7.0 T MRI scan. For in vivo MRI detection of 1×105 SPIO-labelled intraspinally injected CD34+ hUCBCs, the G93A-ALS mouse was scanned at day 0 (injection day) and day 4 to analyse if cell migration could be visualized. At day 0, the injected 1×105 SPIO-labelled cells were clearly detectable due to the SPIO-induced signal extinctions at 7.0 T (Figure 4A). After 4 days, the signal was still clearly detectable, but no migration of the cells was observed (Figure 4B).

Figure 2.
Figure 2.

Ex vivo agar gel phantom detection of superparamagnetic iron oxide (SPIO)-labelled human umbilical cord blood stem cells (hUCBCs) by 1.0 T magnet resonance imaging (MRI). The imaging shows the embedded numbers of hUCB cells. IPC, Positive control (1.0 and 1.5 μl Endorem solution); NC1, cell culture medium, NC2, 5×105 unlabelled cells. Detection limit of SPIO-labelled cells was determined as 2.5×105 by 1.0 T MRI. The negative controls NC1 and NC2 show no signals. The positive control IPC shows strong signal extinction.

Discussion

In the present study, the feasibility of SPIO-labelling of canine mammary epithelial cells and of hUCBCs and their subsequent in vitro and in vivo detection by MRI was assessed. Transplantation of stem or progenitor cells into the CNS is being widely investigated as a novel therapeutic option for a variety of neurological disorders including stroke, spinal cord injury and neurodegenerative diseases such as Parkinson's disease and ALS (9, 26-28). Adult stem cells such as the CD34+ population of hUCBCs, represent promising candidates for cell therapies in general due to their availability, ethical innocuousness, low tumorigenity and high differentiation potential (8).

We have previously shown that intraspinal injection of hUCBCs into the spinal cord prolongs survival, delays deterioration of motor function and prevents motor neuron loss and astrocytosis in mutant SOD1 transgenic mice (13). However, little is known about the long-term differentiation and migration of transplanted stem cells. In experimental studies on transgenic rodent ALS models, different methods for detection of transplanted cells, such as hUCBCs or other types of stem cells, are used. The use of fluorescence-labelled cells (29), specific mRNA expression measurement (10, 29, 30), and immunohistological analysis of dissected sacrificed animals at different time intervals (10, 11, 30) are commonly used techniques. For successful translation into clinical trials, it will however be mandatory to establish non-invasive imaging protocols to better understand potential mechanisms of action of cellular therapies.

We have now developed a protocol for successful in vivo detection by MRI of SPIO-labelled hUCBCs after transplantation into the spinal cord. As the SPIO-labelling efficiency is dependent on the cell type under use, the detection limit of the target cell type was first verified in vitro by scanning agar gel phantoms loaded with SPIO-labelled cells. We determined the lowest detectable cell number of our SPIO-labelled proof-of-concept cell line MTH53SA by 1.0 T and 7.0 T MRI in order to assess the visualization potential of SPIO cell-labelling in general and to examine if the field strength is inversely proportional to the detectable SPIO-labelled cell number. The lowest detectable number of SPIO-MTH53A cells was determined to be 1×105 with 1.0 T (Figure 1A) and 2.5×104 with 7.0 T field strength (Figure 1B). As a 4-fold smaller cell number was detectable at 7.0 T, the comparison of the in vitro 1.0 T and 7.0 T data indicates that the field strength is not directly inversely proportional to the detectable cell number. These data were used as basic concept for hUCBC-labelling and MRI.

Due to the fact that a minimum of 2.5×105 SPIO-labelled hUCBCs was clearly detectable at 1.0 T field strength (Figure 2), the 7.0 T in vitro MR scan of the agar gel phantom was omitted and a cell number of 1×105 implanted hUCBCs was set for 7.0 T in vivo imaging. Taken into account the difference in field strength from 1.0 T to 7.0 T and the thereby available higher resolution at 7.0 T, the detection of 1×105 hUCBCs in vivo was to be expected due to the fact that 2.5×105 hUCBCs are clearly visible at 1.0 T in the agar gel phantom (Figure 2).

Figure 3.
Figure 3.

Prussian blue staining of superparamagnetic iron oxide (SPIO)-labelled canine MTH53A cells (A) and CD34+ human umbilical cord blood stem cells (B), showing positively labelled cells (blue staining) with accumulated iron in the cytosol.

Figure 4.
Figure 4.

In vivo 7.0 T magnet resonance imaging (MRI) scan. Detection of 1×105 SPIO-labelled intraspinally injected hUCB cells in a G93A-ALS mouse. In vivo scans were performed at day 0 (injection day) and day 4. The cells are clearly detectable (arrow), showing the SPIO-induced T2*-specific signal extinctions at the injection site (Figure 4A). Four days post-injection, the signal is still clearly detectable at the injection site (Figure 4B).

Prior to MRI, Prussian blue staining was performed to verify SPIO cell labelling. Figure 3A and B show positively stained MTH53A cells and hUCBCs with accumulated iron in the cytosol. The blue staining demonstrates that the labelling was successful.

Numerous reports are available on in vivo tracking of iron oxide nanoparticle-labelled stem and progenitor cells in cell therapy for various diseases (20, 22, 23, 31-34), but only few studies recently investigated the detection of transplanted SPIO-labelled stem cells in the spinal cord of rodent models, which differed in the used cell type, SPIO particles and MR field strength. On this context, Dunning et al. imaged 5X104 implanted SPIO-labelled Schwann cells and olfactory ensheathing cells after transplantation into focal areas of demyelination in adult rat spinal cords with a 9.4 T MR imager (35). In comparison to our results generated with a lower field strength of 7.0 T, twice the number of implanted cells (1×105) and with regard to the different section planes, we achieved similar intensities of T2-weighted SPIO-induced hypointense signal extinctions.

Another study by Hu et al. examined the fate of 5×104 SPIO-labelled hUC MSCs after transplantation into focal areas in adult rat spinal cord (23). Signal extinctions were detectable for up to 14 days using a 1.5 T MR imager and a T2-weighted protocol, but no migration of the implanted cells was observable. Regarding signal intensity, the results by Hu et al. are in agreement with our finding although lower field strength and half the cell number was used. The signal extinction area of our implanted SPIO-labelled hUCBCs appears somewhat sharper due to the higher field strength MRI with 7.0 T. It should be noted that the labelling efficiency depends on the targeted cell type and the labelling time. The type of superparamagnetic iron oxide nanoparticles used for cell labelling and the MRI sequences are also relevant (36). This might explain why imaging of a lower number of SPIO labelled cells can cause more intensive signal extinctions.

MRI of SPIO-labelled cells in the spinal cord of rodents was also performed by Jendelova et al. (18). In this study, the migration of 2×106 intravenously-injected rat bone marrow stromal cells into the injured spinal cord of Wistar rats in a 4.7 T Bruker tomographer was examined. SPIO-labelled cells were clearly detectable in the spinal cord. Due to the fact that in their study the injection was intravenous and not intraspinal, no information about the number of the imaged cells can be given and a comparison of their data to ours concerning implanted cell numbers is not possible.

To summarize, we were able to successfully detect 1×105 SPIO-labelled intraspinally injected hUCBCs in vivo in a transgenic mouse model of ALS by 7.0 T MRI for the first time. No migration of the injected cells in the form of signal reduction or dislocation was observed along the spinal cord, but a persistent signal was detectable after four days (Figure 4B). Possibly the length of time of four days was not sufficient to monitor cell migration. To investigate this in detail, a longer observation time might be required. In reference to the studies of Dunning et al. (35) and Jendelova et al. (18), a time period of two to four weeks could be adequate. In addition to this, a stimulation of the transplanted cells with special mobilization factors might also improve cell migration.

The major limitation of this study is the dependency of the cell detection limit in relation to the MR field strength. At present, visualization at the single-cell level by 7.0 T MRI is unlikely and will rather require alternative in vivo imaging techniques such as intravital microscopy. In vivo monitoring of cell-cell interactions will, however, be of interest mainly for basic science-related questions. For cellular therapy in animal models and its translation into clinical application, administration of large cell numbers will be required so that the detection limit of MRI does not consist a problem in this context.

Due to the non-invasive character of MRI studies, the nanoparticle-based cell detection further allows to study cell behaviour in single individuals at different stages significantly reducing intra-individual variance and the total number of requested animals.

Further studies could also include a canine model with spontaneously occurring ALS due to an SOD1 missense mutation. In dogs, this spontaneously occurring form of ALS is named degenerative myelopathy. The development of therapeutic options is already of considerable interest for dogs as patients. Moreover, in comparison to the mouse model of ALS, canine degenerative myelopathy closely reflects the human disease regarding the anatomy of the nervous system and the disease course. We therefore now intend to perform further studies using SPIO-labelled canine and human CD34+ cells in mice and dogs over longer time intervals to evaluate the long-term behaviour of intraspinally transplanted cells with respect to signal intensity and cell migration. Ultimately, these data will provide a basis for developing valuable tools for the monitoring of clinical trials assessing the benefit of cell transplantation in humans.

  • Received October 12, 2011.
  • Revision received November 14, 2011.
  • Accepted November 15, 2011.

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In Vivo MRI of Intraspinally Injected SPIO-labelled Human CD34+ Cells in a Transgenic Mouse Model of ALS
S. WILLENBROCK, S. KNIPPENBERG, M. MEIER, R. HASS, P. WEFSTAEDT, I. NOLTE, H. MURUA ESCOBAR, S. PETRI
In Vivo Jan 2012, 26 (1) 31-38;
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